Summary of Work: Inherited mutations of the BRCA1 and BRCA2 genes confer profound predispositions to breast and ovarian cancer in women and hundreds of hereditary alterations have been identified in cancer families. To identify functional domains of BRCA1 and BRCA2 based on evolutionary conservation, we have characterized the mouse and rat homologues of these genes. As observed previously for BRCA1, the rodent homologues of BRCA2 are only 58% identical to the human gene product. The BRCA2 proteins share a potential nuclear localization signal (human codons 3263-3269), the BRC repeat motif, and several highly conserved domains including a large carboxyl region (86% similarity over 674 amino acids). We have generated a HA-expression vector for the wild type rat carboxyl BRCA2 domain and used site-directed mutagenesis to replace the putative nuclear localization signal with Ala residues. Transfections with these constructs are in progress to determine whether BRCA2 localized in the nucleus, as found for BRCA1. In situ hybridization studies indicate that the murine BRCA1 and BRCA2 genes are coordinately regulated in most tissues. To develop mouse models for human breast cancer susceptibility, we have used gene targeting. Phenotypic analyses of BRCA2-deficient mice are underway. Pathologic and neoplastic phenotypes are being followed in hemizigous (+/-) Brca2 mice that carry a neo cassette which disrupts exon 10 and 11. These experiments include low-level radiation exposures (0.25 GY) of BRCA1- and BRCA2-deficient mice to assess carcinogenic risks associated with mammography for women with BRCA1- and BRCA2-defects. Since initial results suggest that the Brca2 (-/-) genotype yields embryonic lethality, we are currently generating a targeting construct based on the Cre-LoxP system that will specifically disrupt BRCA2 exon 27 in the mammary gland during puberty. Crosses with and MMTV-Cre mice (provided by Bev Koller, UNC) will delete BRCA2 exon 27 which contains the putative nuclear localization signal and has been shown to interact with the Rad51 protein in yeast two hybrid assays.